PROJECT SUMMARY Craniofacial anomalies are among the most common and debilitating human birth defects, affecting 1/500 to 1/2000 births depending on the population. Diagnosis and treatment of craniofacial anomalies is further complicated by variation in the severity (phenotypic penetrance) of defects. It is widely recognized that similar genetic mutations often express a spectrum of disease phenotypes, but it is still unknown what mechanisms contribute to this variation. For example, human patients with mutations in Satb2 exhibit a range of craniofacial phenotypes, including small lower jaws (micrognathia) and variable penetrance in cleft palate. Similarly, in mice, Satb2 has a dosage-effect on jaw size. Notably, Satb2 heterozygotes exhibit the full range of jaw size variation between WT (normal) to mutant (severe micrognathia and cleft palate). That such variation in size occurs on an inbred, genetically identical background, suggests that developmental (non-genomic) variation in molecular determinants (e.g., mRNA, protein) contributes to variation in jaw size. Yet, it is unknown what mechanisms cause such variation. Our central hypothesis is that cellular variation in proliferation and apoptosis of mesenchymal progenitors of the jaw results from differences in the levels and variance of Satb2 molecular determinants. Subtle random variation in the production of molecular determinants may affect cell fate when RNA or protein concentrations are near a threshold level (as predicted for Satb2 heterozygotes). Such a threshold model would produce a non-linear relationship between genotype and phenotype, which has previously been predicted to explain high levels of morphological variation in disease models. Using both an in vivo (Aim 1) and in vitro (Aim 2) approach, we will evaluate how levels and variance of Satb2 molecular determinants contribute to variation in the size of the jaw by regulating survival of jaw progenitors. Data generated from work in this proposal will provide essential knowledge about the role of development in generating phenotypic variation. This approach will significantly advance developmental biology by improving methods for relating tissue-level morphology to single-cell molecular biology. Importantly, this work will also substantially enhance the research environment and provide exciting opportunities for undergraduates to conduct high-impact research, preparing them to enter the biomedical workforce.